The present invention relates to the immobilization of enzymes.
Immobilized enzymes are becoming of increased industrial importance, particularly with the upsurge in interest in the subject popularly termed "biotechnology". In fact, immobilized enzymes have long been proposed as an efficient system for carrying out reactions catalysed by enzymes though there have been practical difficulties in achieving economic continuous operation.
To take one example, enzymes have been used in solution for many years for batch hydrolysis and thinning of starches in the production of glucose syrups. There have been many suggestions for ways in which the enzymes could be immobilized to permit continuous operation, but none of these suggestions has met with unqualified acceptance.
Apart from immobilization of enzymes themselves, various techniques have been put forward as ways in which the enzymes could be immobilized without isolation. In particular, whole cells of micro-organisms can be immobilized, thus using the cell as a carrier for the enzyme and obviating the need for extraction of the enzyme from the cell.
Of the immobilization techniques which I have tried for immobilization of cells, I find that immobilization by entrapment within a gel, especially an alginate gel, can give various advantages stemming from the fact that the cells are trapped in an inert, three-dimensional polymer network with relatively large interconnected interstitial spaces in the gel. More generally, other workers have reported on the usefulness of gel entrapment, especially entrapment in an alginate gel, as an immobilization technique.
One problem with gel-immobilized cells is a marked tendency for activity to be lost during storage or other periods of non-use, for instance during transportation. An accompanying difficulty during non-use is the tendency for contaminating micro-organisms to proliferate. It is a relatively routine matter to prepare gel-immobilized cells which have high activity upon immediate use, but the activity tends to decay relative quickly if the gel-immobilized cells are not used. The gel has a high water activity and probably provides a good environment for growth of contaminant moulds, bacteria and the like.
Operational stability and storage stability are useful concepts when reviewing the extent of any activity loss.
During operation of a conversion process using gel-immobilized cells, there is usually some progressive loss in the activity of the enzyme system. This decay can be slow, particularly if the cells are operating on a nutritionally deficieint medium which is inadequate for sustaining microbial growth. The loss in the activity can be assessed as a half-life, in the same way as radioactive decay. For instance, it might be that the activity of gel-immobilized cells is reduced by half during a period of use of say 2000 hours.
In a similar manner, it is possible to monitor any loss of activity while the gel-immobilized cells are not in use. Measurement of the activity before and after storage or other inactive period gives the activity loss, and the half-life obtained in this way can be called the storage half-life. In general, this storage half-life is less than the operational half-life.
Thus, it is often found that when freshly prepared gel-immobilized cells with high operational stability are not put into immediate use, the operational stability is much less at the time when the cells are eventually put into use. If gel-immobilized cells are stored at low temperatures, it sometimes adequate to store them in bottles covered with plastics wrap-film, but at higher (ambient) temperatures where fungal spores might be more plentiful this procedure is normally insufficient. Microbial growth, chiefly moulds, is typically observed. Consequently, the cells have to be stored in gas-tight tubes and only opened immediately before use. It will be appreciated that even when this mode of storage is successful, it is not a practical proposition for large-scale industrial use.
We have examined ways in which the immobilized cells could be stored, particularly with a view to developing a storage method which allows easy handling and transportation of the immobilized enzyme system. The conventional methods of preserving biological materials in the food industry include irradiation, dehydration, chilling, or the use of extremes of pH or osmotic pressure. However it is preferable to avoid excessive exposure to stresses such as extreme of temperature or pH, osmotic shock, and in particular composite stresses such as combinations of the above stresses, especially if a high storage stability is desired.
To give one specific example, freeze-drying frequently leads to an appreciable loss in activity and a short operational and storage half-life.